There has been increasing demand in the research reagent, diagnostic reagent and chemical process industries for protein-based catalysts possessing novel capabilities. At present, this need is largely addressed using enzymes purified from a variety of cultivated bacteria or fungi. However, because less than 1% of naturally occurring microbes can be grown in pure culture (Amann, 1995), alternative techniques must be developed to exploit the full breadth of microbial diversity for potentially valuable new products.
Virtually all of the commercial enzymes now in use have come from cultured organisms. Most of these organisms are bacteria or fungi. Amann et al. (Amann, 1995) have estimated cultivated microorganisms in the environment as follows:
______________________________________ Habitat Culturability (%) ______________________________________ Seawater 0.001-0.1 Freshwater 0.25 Mesotrophic lake 0.01-1.0 Unpolluted esturine waters 0.1-3.0 Activated sludge 1.0-15.0 Sediments 0.25 Soil 0.3 ______________________________________
These data were determined from published information regarding the number of cultivated microorganisms derived from the various habitats indicated.
Other studies have also demonstrated that cultivated organisms comprise only a small fraction of the biomass present in the environment. For example, one group of workers recently reported the collection of water and sediment samples from the "Obsidian Pool" in Yellowstone National Park (Barns, 1994) where they found cells hybridizing to archaea-specific probes in 55% of 75 enrichment cultures. Amplification and cloning of 16S rRNA encoding sequences revealed mostly unique sequences with little or no representation of the organisms which had previously been cultured from this pool, suggesting the existence of substantial diversity of archaea with so far unknown morphological, physiological and biochemical features. Another group performed similar studies on the cyanobacterial mat of Octopus Spring in Yellowstone Park and came to the same conclusion; namely, tremendous uncultured diversity exists (Ward, 1990). Giovannoni et al. (1990) and Torsvik et al. (1990a) have reported similar results using bacterioplankton collected in the Sargasso Sea and in soil samples, respectively. These results indicate that the exclusive use of cultured organisms in screening for useful enzymatic or other bioactivities severely limits the sampling of the potential diversity in existence.
Screening of gene libraries from cultured samples has already proven valuable. It has recently been made clear, however, that the use of only cultured organisms for library generation limits access to the diversity of nature. The uncultivated organisms present in the environment, and/or enzymes or other bioactivities derived thereof, may be useful in industrial processes. The cultivation of each organism represented in any given environmental sample would require significant time and effort. It has been estimated that in a rich sample of soil, more than 10,000 different species can be present. It is apparent that attempting to individually cultivate each of these species would be a cumbersome task. Therefore, novel methods of efficiently accessing the diversity present in the environment are highly desirable.